Gai et al. Malar J (2019) 18:328 https://doi.org/10.1186/s12936-019-2966-9 Malaria Journal

RESEARCH Open Access Dufy antigen receptor for chemokines gene polymorphisms and malaria in Mangaluru, India Prabhanjan P. Gai1*, Welmoed van Loon1, Konrad Siegert1, Jakob Wedam1, Suyamindra S. Kulkarni2, Rashmi Rasalkar2, Archith Boloor3,4, Arun Kumar5, Animesh Jain3,4, Chakrapani Mahabala3,4, Shantaram Baliga3,4, Rajeshwari Devi6, Damodara Shenoy3,4, Pramod Gai2 and Frank P. Mockenhaupt1

Abstract Background: Dufy blood group antigens serve as receptors for Plasmodium vivax invasion into erythrocytes, and they are determined by polymorphisms of the Dufy antigen receptor for chemokines (DARC), also known as Fy glycoprotein (FY). Dufy negativity, i.e., absence of the antigens, protects against P. vivax infection and is rare among non-African populations. However, data on DARC​ polymorphisms and their impact on Plasmodium infection in India are scarce. Methods: In a case–control study among 909 malaria patients and 909 healthy community controls in Mangaluru, southwestern India, DARC​ polymorphisms T-33C (rs2814778), G125A (rs12075), C265T (rs34599082), and G298A (rs13962) were genotyped. Associations of the polymorphisms with the odds of malaria, parasite species and manifes- tation were assessed. Results: Among patients, vivax malaria (70%) predominated over falciparum malaria (9%) and mixed species infec- tions (21%). DARC ​T-33C was absent and C265T was rare (1%). FYB carriage (deduced from DARC ​G125A) was not associated with the risk of malaria per se but it protected against severe falciparum malaria (P 0.03), and hospitaliza- tion (P 0.006) due to falciparum malaria. Vice versa, carriage of DARC​ 298A was associated with= increased odds of malaria= (aOR, 1.46 (1.07–1.99), P 0.015) and vivax malaria (aOR, 1.60 (1.14–2.22), P 0.006) and with several reported symptoms and fndings of the patients.= = Conclusion: This report from southern India is the frst to show an independent efect of the DARC ​298A polymor- phism on the risk of malaria. Functional studies are required to understand the underlying mechanism. Moreover, FYB carriage appears to protect against severe falciparum malaria in southern India. Keywords: Dufy, DARC​, SNPs, Malaria, India, Plasmodium vivax, Plasmodium falciparum

Background and/or its manifestation [2, 3]. Tis includes the Dufy Malaria is considered a major driving force in shaping antigen receptor for chemokines (DARC, or Dufy anti- the human genome [1]. “Classical” erythrocyte variants gen), which is a glycosylated erythrocyte membrane pro- such as the sickle-cell trait ofer relative resistance against tein. Te encoding DARC​ gene is located on chromosome malaria and are thus subject to evolutionary selection in 1. A common DARC ​ polymorphism, G125A (rs12075), endemic regions. In addition, various further host genetic generates the FYA (G125) and FYB (125A) alleles. Te polymorphisms infuence susceptibility to the disease resulting genotypes include the wildtype FYA/FYA, which correspond to the phenotype Fy (a+, b−), FYA/ FYB (Fy (a , b )) and FYB/FYB (Fy (a , b )). An addi- *Correspondence: [email protected] + + − + 1 Charité-Universitätsmedizin Berlin, corporate member of Freie tional T-33C mutation silences antigen expression giving Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute rise to Dufy blood group negativity (Fy (a , b )). Fur- of Health; Institute of Tropical Medicine and International Health, Berlin, − − Germany ther single nucleotide polymorphisms (SNPs), C265T Full list of author information is available at the end of the article and G298A, are together responsible for weakening the

© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gai et al. Malar J (2019) 18:328 Page 2 of 8

expression of the FYB allele, whereas G298A alone is not in each of the 60 census wards of Mangaluru yielding a able induce this efect [4]. total number of 2478 individuals. Te study protocol was In line with its function as a multi-specifc receptor approved by the Institutional Ethics Committee of Kas- for a wide range of chemokines [5, 6], the absence of turba Medical College, Mangalore, Manipal University, DARC on the erythrocyte cell surface (Dufy blood group and permission to conduct the study was granted by the negativity) has been associated with diverse conditions Directorate of Health and Family Welfare Services, Gov- including infammation, HIV infection, and malignan- ernment of Karnataka. Informed written consent was cies [7, 8]. With respect to malaria, Dufy blood group obtained from all individuals enrolled in this study. negativity is predominant among Africans and renders Details of the patient recruitment process as well as erythrocytes resistant to invasion by Plasmodium vivax socio-economic, clinical and laboratory data have been and Plasmodium knowlesi [5, 9–12]. Moreover, binding reported elsewhere [22]. Briefy, socio-economic data of DARC to platelet factor 4 (PF4) is essential for plate- were collected by trained interviewers from patients let-mediated killing of Plasmodium falciparum parasites (cases) and controls. Venous blood was collected into [13, 14]. Associations of DARC​ genotypes with vivax EDTA from malaria patients and by fnger prick blood malaria are reportedly conficting. For instance in Bra- on Whatman™ 3MM paper from controls. Malaria para- zil, FYA/FYA conferred reduced odds of vivax malaria sites were counted per 200 white blood cells (WBCs) on [15]. However, in another Brazilian study, FYA/FYA was Giemsa-stained thick blood flms, and parasite species signifcantly more frequent in vivax malaria patients as was defned based on thin-flm microscopy. Following compared to healthy blood donors without a history of DNA extraction (QIAamp DNA Blood Mini kit, Qia- malaria [11]. In India, the FYA allele has been associated gen, Hilden, Germany), Plasmodium species was ascer- with a reduced incidence of vivax malaria and the FYB tained by semi-nested polymerase chain reaction (PCR) allele with an increased one [16]. However, the few avail- assays [23]. Out of 2383 Plasmodium-negative controls, able individual studies from India did not show a link 909 were randomly selected for this case–control study. between DARC​ genotypes and vivax malaria [17–19]. DARC​ SNP genotyping including T-33C, G125A, C265T In India, FYA/FYA is the predominant genotype and and G298A was achieved by melting curve analysis on Dufy negativity occurs only in a few tribal populations the Light Cycler 480 instrument (Roche, Basel, Switzer- [16, 19]. At the same time, India contributes to nearly half land) using commercial primers and probes; reagent con- of the global Plasmodium vivax cases, and P. vivax and centrations and PCR conditions are available with the P. falciparum are responsible for 37% and 63% of malaria manufacturer (TIB MOLBIOL, Berlin, Germany). cases, respectively [20]. Tis provides the opportunity Data analysis was performed using RStudio 3.5.1 (2018) to study the efect of DARC​ genotypes on the risk of (Integrated Development for R. RStudio, Inc., Boston, malaria per se, and of vivax and falciparum malaria sepa- USA) and SPSS 25 (IBM Corp., Armonk, USA). Te rately. Of note, the manifestation of Plasmodium infec- distribution of DARC ​genotypes between case and con- tion is not only caused by the infecting parasite but also trols were compared by χ2 test or Fisher’s exact test as by pro-infammatory host responses, which potentially appropriate, and odds ratios (ORs) and 95% confdence contribute to pathophysiology [21]. In this regard, the intervals (95% CI) were calculated. Binomial logistic function of DARC as a receptor for diverse chemokines regression was used to calculate the adjusted odds ratios [5, 6] might possibly infuence the clinical manifesta- (aORs) of individuals with variant genotypes for malaria tion. Against this background, the present study aimed per se and for malaria separated by species with prob- at describing the DARC​ genotype distribution pattern in able confounders: age, sex and migration to Mangaluru. Mangaluru and as a next step at examining the associa- Continuous parameters were compared using Student’s tion of DARC​ genotypes with (i) malaria, (ii) malaria as t test, Mann–Whitney U test, or Kruskal–Wallis test as caused by the various Plasmodium spp., and (iii) clinical applicable. A P value < 0.05 was considered statistically presentation. signifcant.

Methods Results A total of 909 malaria out-patients were recruited at Essential characteristics of malaria patients and con- Wenlock Hospital, Mangaluru, Karnataka, India between trols are displayed in Table 1. More than 90% of malaria June to December 2015. Wenlock Hospital (900 beds) is patients were male adults. Teir median age was the largest governmental hospital in Mangaluru ofer- 26 years, and more than three in four had migrated to ing treatment particularly for the economically-deprived Mangaluru a median period of 6 months before presen- part of the population. In parallel, an average of 40 (26– tation (range 1–600 days). Teir overall socio-economic 53) healthy community controls were randomly recruited status including educational background was low, and Gai et al. Malar J (2019) 18:328 Page 3 of 8

Table 1 Characteristics of malaria patients and controls Parameter Cases Controls P

No. 909 909 Male gender (%, n) 92.8 (844) 57.5 (523) < 0.0001 Age (years; median, range) 26 (4–82) 30 (1–94) 0.0001 Migration (%, n) 77.8 (706/907) 34.4 (313/909) < 0.0001 Socio-economic parameters No formal education (%, n) 33.0 (298/902) 11.1 (98/882) < 0.0001 Occupation as construction worker or daily labourer (%) 56.1 36.3 < 0.0001 Monthly family income (rupees; median, range), cases; n 893, 6000 (0–35,000) 7000 (500–100,000) 0.06 controls; n 575 = = Stated use of a bed net in preceding night (%, n) 39.1 (354,906) 54.3 (484/892) < 0.0001 Stated use of a window net (%, n) 4.2 (38/906) 42.4 (376/890) < 0.0001 Presence of stagnant water bodies (%, n) 31 (281/906) 3.3 (29/851) < 0.0001 Plasmodium prevalence P. vivax 69.6 (633) 0 – P. falciparum 9.0 (82) 0 – P. vivax and P. falciparum mixed 21.3 (194) 0 – Geometric mean parasite density (/µl; 95% CI) All patients 3412 (3081–3779) – – P. vivax 2999 (2660–3382) – – P. falciparum 5408 (3758–7750) – – P. vivax and P. falciparum mixed 4246 (3413–5283) – – DARC​ G125A genotypes (%) GG (FYA/FYA) 43.9 43.1 1 GA (FYA/FYB) 44.1 43.7 0.91 AA (FYB/FYB) 11.9 13.1 0.48 GA or AA (FYB carriers) 56.1 56.8 0.74 DARC​ G298A genotypes (%) GG 83.3 85.5 1 GA 15.6 13.3 0.16 AA 1.1 1.2 0.87 GA or AA 16.7 14.5 0.19

more than half of the patients were either construc- (P < 0.0001). In the study sample and based on DARC​ tion workers or daily labourers [22]. In comparison, G125A, FYA/FYB (43.9%) and FYA/FYA (43.5%) were among control individuals, the proportion of males the most common Dufy genotypes (FYB/FYB, 12.5%). was lower, age was higher, and only a minority had Of note, these genotypes did not difer between cases migrated to Mangaluru city (each, P < 0.0005). Among and controls, and thus were not associated with the patients, vivax malaria (70%) predominated over falci- odds of malaria (Table 1), irrespective of stratifcation parum malaria (9%), and mixed P. vivax–P. falciparum by parasite species (Table 2). infections (21%). Te geometric mean parasite density In contrast, carriage of DARC​ 298A (GA or AA), i.e., (GMPD) was 3412/µl (95% CI 3081–3779). genotypes involved in but not solely responsible for a Te DARC​ SNP-33 T>C was absent in 570 random weakened expression of the FYB allele [4], appeared to samples genotyped, and the SNP 265 C>T was rare be more common in malaria patients (16.7%, 152/909; (1% (6/564) heterozygous). Tese polymorphisms were P = 0.19) and in vivax malaria patients in particu- thus omitted from analysis. Genotyping of the DARC​ lar (17.7%, 112/633; P = 0.09) as compared to controls SNPs G125A and G298A was successful in all patients (14.5%, 132/909) (Table 2; Fig. 1). Adjusting for the and controls. DARC​ 298A occurred exclusively when observed diferences in age, sex, and migration, carriage also 125A was present, i.e., on an FYB background of DARC​ 298A was associated with increased odds of Gai et al. Malar J (2019) 18:328 Page 4 of 8

Table 2 Genotype distribution among malaria patients and controls separated by Plasmodium species SNP Genotype Controls, n 909 Malaria patients = P. vivax, n 633 P. falciparum, n 82 P. vivax and P. = = falciparum, n 194 = DARC​ G125A (n, %) GG (FYA/FYA) 392 (43.1) 280 (44.2) 36 (43.9) 83 (42.7) GA (FYA/FYB) 398 (43.7) 277 (43.7) 34 (41.4) 90 (46.3) AA (FYB/FYB) 119 (13.1) 76 (12.0) 12 (14.6) 21 (10.8) GA or AA (FYB carriers) 517 (56.8) 353 (55.7) 46 (56.1) 111 (57.2) DARC​ G298A (n, %) GG 777 (85.5) 521 (82.3) 69 (84.2) 167 (86.1) GA 121 (13.3) 105 (16.6) 11 (13.4) 26 (13.4) AA 11 (1.2) 7 (1.1) 2 (2.4) 1 (0.5) GA or AA 132 (14.5) 112 (17.7) 13 (15.8) 27 (13.9)

Fig. 1 DARC​ 298A carriage is associated with increased odds of malaria and vivax malaria. OR, odds ratio; 95% CI, 95% confdence interval; unadjusted, crude odds ratio; adjusted, odds ratio adjusted for age, sex, and migration status; *P < 0.05 as compared to DARC​ G298 wildtype individuals. Forest plots display the odds of malaria according to DARC​ G298A genotypes. χ2 test or Fisher’s exact test was applied to calculate unadjusted odds ratios (95% CIs). Adjusted odds ratios were derived from logistic regression models adjusting for age, sex and migration status. As compared to control individuals (14.5%, 132/909), DARC​ 298A carriage was more common in malaria patients [16.7%, 152/909; aOR, 1.46 (1.07–1.99)] and in vivax malaria patients in particular [17.7%, 112/633; aOR, 1.60 (1.14–2.22)]

highest in individuals with FYA/FYA (5.0%, 20/399), malaria (aOR, 1.46 (1.07–1.99), P = 0.015) and of vivax lower in FYA/FYB (3.5%, 14/401, P 0.29) and low- malaria in particular (aOR, 1.60 (1.14–2.22), P = 0.006) = (Fig. 1). No signifcant association with falciparum or est in FYB/FYB (0.9%, 1/109, P = 0.06). Tis was due mixed species malaria was observed. to the absence of hospital admissions in patients with In a next step of analysis among malaria patients, the falciparum malaria carrying the FYB allele (P = 0.006, proportions of hospitalization and of severe malaria Table 3). Severe malaria due to any parasite species were compared between DARC​ genotypes. For that, occurred at similar proportions in patients with the dif- FYA/FYA and wildtype DARC​ G298, respectively, ferent FY genotypes, but severe falciparum malaria was were set as reference groups. Among the patients, 3.5% absent in individuals carrying FYB (P = 0.03, Table 3). (32/909) and 3.8% (35/909) of individuals were hospi- DARC​ 298A carriage did neither afect the propor- talized and had severe malaria, respectively. Te pro- tion of hospitalized patients nor that of severe malaria. portion of patients who were admitted to ward was Also, it did not substantially change the associations of Gai et al. Malar J (2019) 18:328 Page 5 of 8

Table 3 Proportion of patients with hospitalization and severe malaria according to FY genotypes FYA/FYA FYA/FYB FYB/FYB FYB carriage with DARC​ 298A carriage

Hospitalizationa All species 5.0% (20/399) 3.5% (14/401) 0.9% (1/109) 2.6% (4/152) P. vivax 3.6% (10/280) 3.6% (10/277) 1.31% (1/76) 3.6% (4/112) P. falciparum 16.7% (6/36) 0% (0/34)* 0% (0/12) 0% (0/13) Mixed 4.8% (4/83) 4.4% (4/90) 0% (0/21) 0% (0/27) Severe ­malariab All species 3.5% (14/399) 3.7% (15/401) 2.8% (3/109) 3.3% (5/152) P. vivax 2.9% (8/280) 3.6% (10/277) 2.6% (2/76) 3.6% (4/112) P. falciparum 11.1% (4/36) 0% (0/34) 0% (0/12) 0% (0/13) Mixed 2.4% (2/83) 5.6% (5/90) 4.8% (1/21) 3.7% (1/27) a Of 35 patients admitted to ward, 10 were categorized as severe malaria patients. Other reasons included vomiting (5), dehydration (2), co-morbidities (2), weakness (2), suspected typhoid fever (1), jaundice (1), recent delivery (1), patient request (1), low blood pressure (1) as well as retrospectively not ascertainable causes (9) b 32 patients had severe malaria according to the WHO defnition, i.e., hypotension (15; impaired perfusion not assessed), renal impairment (5), renal impairment and respiratory distress (1), severe malarial anaemia (4), prostration (3), confusion (2), jaundice (1), and abnormal bleeding (haematemesis, 1). Impaired consciousness, convulsions, hypoglycaemia, acidosis, hyperparasitaemia and pulmonary oedema were not observed * P < 0.05 as compared to FYA/FYA

FYB carriage with the odds of hospitalization or severe which risk estimates had to be adjusted accordingly. Te malaria (Table 3). DARC​ polymorphisms T-33C and C265T were too rare Lastly, signs and symptoms as well as laboratory to deduce meaningful fndings. No interaction in terms of parameters were analysed with respect to DARC ​ geno- associations with malaria or signs or symptoms was seen types. Tese did not difer signifcantly with the three for the FYA or FYB alleles and DARC​ G298A. Terefore, FY genotypes. However, DARC​ 298A carriage was asso- data were presented separately. ciated with increased proportions of patients reporting In the present study, the FYA/FYA and FYA/FYB geno- a history of muscle pain, back pain, fatigue, and at bor- types occurred in each approximately 44%. Among more derline, diarrhoea. Basically, the same fndings were seen than 3000 blood donors in New Delhi, FYA/FYA and for vivax malaria, whereas in falciparum malaria DARC​ FYA/FYB were observed in 32.5% and 48.9%, respec- 298A carriage was associated with a history of sweats tively. Dufy blood group negativity, absent in the present (P = 0.05) and of vomiting (Table 4). Clinically, the pro- study, was observed in 0.3% [24]. Of note, the proportion portions of splenomegaly and of elevated bilirubin con- of Dufy blood group genotypes difers across India but centration were increased in patients with DARC​ 298A fndings from the South of the country closely match with carriage as was axillary temperature (P = 0.05), specif- the prevalence data of the present study [16]. In com- cally in mixed species infections (P = 0.01). parison to other ethnic groups, the predominant FYA/ FYB genotype in the present study is slightly more com- Discussion mon in Caucasians (49%) but rare in sub-Saharan Afri- Te present results indicate that FYB carriage in an cans (1.0%) and Chinese (8.9%). Tis is due to a higher Indian population does not infuence the risk of malaria FYA allele frequency among Indians than in Caucasians per se, but, in case of P. falciparum infection, it is asso- and sub-Saharan Africans. Dufy blood group negativity ciated with protection from hospitalization and severe is absent or very rare in all populations except for sub- malaria. Vice versa, DARC ​ 298A carriage appeared to Saharan Africans (68%) [24]. Carriage of DARC​ 298A increase the risk of malaria, and of vivax malaria in par- was found in 15.6% of the current study participants, cor- ticular, and to afect the occurrence of several symptoms. responding to an allele frequency of 0.08. Based on the Despite its sample size the study has several limita- 1000 Genomes Project, this matches the respective fgure tions which need to be considered when interpreting the of 0.09 among South Asians, but it is lower than the allele results: subgroups, e.g., patients with falciparum malaria, frequency of 0.18 among Caucasians and higher than the were relatively small afecting the power of analyses. value of 0.005 in Africans [4]. Patients and controls difered in essential parameters Dufy blood group antigens are known to play an such as age, gender, and migration status, because of important role in P. vivax malaria [5, 6, 25] and to be Gai et al. Malar J (2019) 18:328 Page 6 of 8

Table 4 Patient history as well as signs and symptoms according to DARC​ 298A carriage and parasite species All malaria patients Vivax malaria Falciparum malaria Mixed species malaria Wildtype DARC​ 298A Wildtype DARC​ 298A Wildtype DARC​ 298A Wildtype DARC​ 298A carriage carriage carriage carriage

No 757 152 521 112 69 13 167 27 Reported signs and symptoms in preceding 2 weeks Fever 99.6 (753/756) 100.0 (152) 99.6 (518/520) 100.0 (112) 100.0 (69) 100.0 (13) 99.4 (166) 100.0 (27) Headache 94.0 (711/756) 94.7 (144) 93.1 (484/520) 95.5 (107) 97.1 (67) 92.3 (12) 95.8 (160) 92.6 (25) Muscle pain 84.9 (642/756) 91.4 (139)* 84.2 (438/520) 92.0 (103)* 89.9 (62) 84.6 (11) 85.0 (142) 92.6 (25) Back pain 69.6 (526/756) 77.6 (118)* 67.9 (353/520) 77.7 (87)* 72.5 (50) 69.2 (9) 73.7 (123) 81.5 (22) Fatigue/weak- 86.1 (651/756) 92.8 (141)* 85.0 (442/520) 91.1 (102) 89.9 (62) 92.3 (12) 88.0 (147) 100.0 (27) ness Chills/shivering 88.1 (666/756) 90.1 (137) 88.1 (458/520) 90.2 (101) 82.6 (57) 84.6 (11) 90.4 (151) 92.6 (25) Sweats 73.5 (554/754) 77.6 (118) 76.1 (395/519) 73.2 (82) 65.2 (45) 92.3 (12)* 68.7 (114/166) 88.9 (24)* Cough 42.2 (319/756) 45.4 (69) 41.9 (218/520) 47.3 (53) 43.5 (30) 46.2 (6) 42.5 (71) 37.0 (10) Nausea 41.2 (311/754) 44.1 (67) 40.8 (212/519) 42.0 (47) 42.0 (29) 38.5 (5) 42.2 (70/166) 55.6 (15) Vomiting 29.8 (225/756) 29.6 (45) 28.5 (148/520) 22.3 (25) 30.4 (21) 69.2 (9)* 33.5 (56) 40.7 (11) Diarrhoea 3.3 (25/756) 6.6 (10) 2.9 (15/520) 8.9 (10)* 5.8 (4) 0.0 (0) 3.6 (6) 0.0 (0) Abdominal 31.7 (240/756) 29.6 (45) 30.0 (156/520) 32.1 (36) 40.6 (28) 23.1 (3) 33.5 (56) 22.2 (6) pain Assessed parameters GMPD (/µl; 3364 3641 2995 3036 4681 11,580 4216 4428 95% CI) (3011–3757) (2800–4734) (2626–3415) (2253–4092) (3148–6961) (5055–26,527) (3340–5321) (2347–8354) Splenomegaly 16.8 (126/751) 27.0 (41)* 14.5 (75/516) 25.9 (29)* 27.9 (19/68) 38.5 (5) 19.2 (32) 25.9 (7) (%, n) Axillary tem- 37.2 1.6 37.4 1.5 37.1 1.6 37.3 1.4 37.3 1.7 37.4 2.0 37.3 1.5 38.1 1.4* perature (°C, ± ± ± ± ± ± ± ± mean SD), n 903± = Body mass 19.5 (12.2– 19.3 (13.6– 19.8 (12.3– 19.3 (13.6– 19.1 (12.6– 19.7 (14.6– 19.0 (12.2– 19.3 (16.7–31.9) index (BMI) 46.3) 33.3) 39.5) 33.3) 27.5) 24.9) 46.3) (kg/m2; median, range), n 887 = Hypotension 1.6 (12/756) 2.0 (3) 1.0 (5/520) 1.8 (2) 1.4 (1) 0.0 (0) 3.6 (6) 3.7 (1) (systolic BP < 80 mm Hg; %, n) Severe throm- 12.3 (88/715) 12.5 (18/144) 10.5 (52/495) 13.0 (14/108) 21.0 (13/62) 9.1 (1/11) 14.6 (23/158) 12.0 (3/25) bocytopenia (< 50,000/µl; %, n) Anaemia (%, n) 34.3 (248/722) 35.4 (52/147) 30.1 (151/501) 33.9 (37/109) 41.0 (25/61) 41.7 (5/12) 45.0 (72/160) 38.5 (10/26) Increased 3.3 (23/688) 1.4 (2/140) 2.7 (13/473) 1.0 (1/103) 4.9 (3/61) 8.3 (1/12) 4.5 (7/154) 0.0 (0/25) creatinine (> 1.4 mg/dl; %, n) Increased 46.8 (324/693) 56.1 (78/139)* 46.3 (221/477) 57.3 (59/103)* 53.3 (32/60) 50.0 (6/12) 45.5 (71/156) 54.2 (13/24) bilirubin (> 1.2 mg/dl; %, n)

* P < 0.05 as compared to the wildtype DARC​ G298 essential in platelet-mediated killing of P. falciparum [13]. study FYA or FYB did not afect the odds of malaria, irre- However, actual fndings in various populations including spective of parasite species. Indians have been ambiguous [11, 15–19]. In the present Gai et al. Malar J (2019) 18:328 Page 7 of 8

Tis contrasts with recent report on protective efects with this wide-range receptor function, DARC per se of FYA/FYA against vivax malaria in Brazil [15]. Like- has been associated with several infammatory and wise, in one study from India, FYA was found to be asso- infectious diseases including increased rates of pros- ciated with a reduced 5 years average incidence of vivax tate cancer and asthma as well as an increased risk of malaria [16]. In Brazil, no association with falciparum HIV infection in its absence [7, 27]. DARC also infu- malaria was observed [15], in India, falciparum malaria ences infammation in terms of chemokine levels and was not analysed [16]. In another study from Brazil, FYA/ leukocyte trafcking and malignancy [8]. Monocytes FYA was associated with increased susceptibility to vivax and neutrophils phagocytize infected red blood cells, malaria [11], and in older work from India, no impact of and they are important sources of cytokines, which act the Dufy blood group genotypes on vivax or falciparum as signaling molecules in activating immune responses malaria was observed [17–19]. Te reason for these con- against malaria [28]. Increased phagocytic activity via ficting results may be related to variable proportions of neutrophils is observed in vivax malaria [29]. A pos- P. vivax and P. falciparum among the patients included, sible explanation in support of the present study fnd- partially low sample sizes and genetic variation among ings could be the involvement of variant DARC​ 298A the diverse populations, including the Indian one. Of in altering the chemoattractant properties of the Dufy note, in the present study, falciparum malaria patients glycoprotein, leading to a modifed activation of the with FYA/FYA showed the highest rate of hospitalization pro-infammatory signal cascade. Functional studies and severe malaria, which was unexpected considering are needed to verify this hypothesis. the protective efects against vivax malaria mentioned above [15, 16]. In-vitro, binding of Dufy antigens to platelet factor 4 (PF4) is crucial for platelet-mediated Conclusion killing of P. falciparum [13, 14] even though the role of Tis study from southern India is the frst to show an FY variation in that is unknown. One explanation for independent efect of DARC ​298A in Plasmodium infec- the fnding of reduced odds of severe malaria in patients tion. DARC​ 298A carriage appears to be associated with with FYB carriage could be that it afects binding afnity increased susceptibility to malaria and to vivax malaria towards PF4 and thereby the capacity of platelet medi- in particular, and to worsen several signs and symptoms. ated killing. On the other hand, parasite densities and Functional studies on the role of this polymorphism are other severity markers of infection were not reduced in required to disentangle the underlying mechanisms. Te patients with FYB carriage. Consequently, further work is same applies to the role of FYB genotype carriage pro- required to explain the observed association of FYB car- tecting against severe falciparum malaria. Considering riage with hospitalization and severe falciparum malaria. Dufy blood group antigens being studied as vaccine can- A novel fnding is that carriage of the DARC​ 298A didates against vivax malaria [30] and the present clin- variant increased the odds of malaria by roughly 50%. ico-epidemiological fndings, unravelling the molecular Moreover, this polymorphism was associated with mechanisms of Dufy blood group antigens infuencing increased proportions of patients reporting several malaria susceptibility and resistance is urgently needed. signs and symptoms. Tis SNP has not been observed to be independently associated with malaria. One Abbreviations previous study from Brazil did not observe an asso- DARC​: Dufy antigen receptor for chemokines; FY: Fy glycoprotein; P. vivax: ciation with malaria susceptibility when combining Plasmodium vivax; P. falciparum: Plasmodium falciparum; WBCs: white blood cells; PF4: platelet factor 4; SNP: single nucleotide polymorphism; PCR: DARC​ C265T and G298A as a condition weakening polymerase chain reaction; ORs: odds ratios; CI: confdence interval; GMPD: the expression of Dufy antigens (FYX) [11]. DARC​ geometric mean parasite density; BMI: body mass index. C265T was absent in the present study population. Acknowledgements DARC​ 298 G>A results in an amino acid substitution We thank the patients, staf as well as doctors and administration at Wenlock in the frst intracellular loop of the Dufy glycoprotein. Hospital and Kasturba Medical College, Mangalore. We also thank the control It has been linked with reduced FYB expression only individuals from Mangaluru, staf as well as feld workers of the Mangaluru City Corporation and the District Vector Borne Disease Control Programme Ofce. in the presence of C265T [4, 26]. On the other hand, This work forms part of the doctoral thesis of PPG, WVL, KS and JW. DARC acts as a multi-specifc receptor for chemokines. Tese include the melanoma growth stimulatory activ- Authors’ contributions PPG, FPM, PG, AJ, RD and DS designed the study. PPG, KS, JW, AB, AK, SB, ity, interleukin-8, regulated upon activation normal RD, and DS were responsible for patient recruitment, clinical and laboratory T-expressed, monocyte chemotactic protein-1, neu- examinations. PPG, KS, RR and SK did the PCR analyses. PPG, WVL and FPM did trophil activating protein 2 and 3, epithelial neutrophil the statistical analyses and wrote the paper with major contributions of the activating peptide-78, angiogenesis-related platelet other authors. All authors read and approved the fnal manuscript. factor 1, and growth-related gene alpha [5, 6]. In line Gai et al. Malar J (2019) 18:328 Page 8 of 8

Funding 10. Howes RE, Patil AP, Piel FB, Nyangiri OA, Kabaria CW, Gething PW, This study was supported by grants from the German Research Foundation et al. The global distribution of the Dufy blood group. Nat Commun. (GRK 1673 to PPG and GRK 2046 to WVL) and from the Sonnenfeld-Foundation 2011;2:266. to PPG. The funding bodies had no role in the design of the study and collec- 11. Albuquerque SRL, De Oliveira Cavalcante F, Sanguino EC, Tezza L, Castilho tion, analysis, and interpretation of data and in writing the manuscript. F, Lilian C, et al. FY polymorphisms and vivax malaria in inhabitants of Amazonas State, Brazil. Parasitol Res. 2010;106:1049–53. Availability of data and materials 12. Iwamoto S, Li J, Sugimoto N, Okuda H, Kajii E. Characterization of the The dataset generated and/or analysed in this study is not publicly available Dufy gene promotor: evidence for tissue-specifc abolishment of expres- due to issues of confdentiality and ongoing analyses, but are available from sion in Fy(a-b-) of black individuals. Biochem Biophys Res Commun. the corresponding author on reasonable request. 1996;222:852–9. 13. McMorran BJ, Wieczorski L, Drysdale KE, Chan JA, Huang HM, Smith C, Ethics approval and consent to participate et al. Platelet factor 4 and dufy antigen required for platelet killing of The study protocol was reviewed and approved by the Institutional Ethics Plasmodium falciparum. Science. 2012;338:1348–51. Committee of Kasturba Medical College, Mangalore, Manipal University (IEC 14. McMorran BJ, Burgio G, Foote SJ. New insights into the protective power KMC MLR 05-1598), and informed written consent was obtained by all study of platelets in malaria infection. Commun Integr Biol. 2013;6:e23653. patients. 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